Hepatocyte growth factor (HGF), also known as scatter factor, is a member of the plasminogen-related growth factor family and is a key mediator of cell migration, proliferation, survival, motility and morphogenesis (Stoker, M. et al. (1987) Nature 327: 239-42; Nakamura, T. et al. (1989) Nature 342: 440-3; Bussolino, F. et al. (1992) J Cell Biol 119: 629-41). HGF is known to specifically activate the Met receptor tyrosine kinase, resulting in downstream activation of RAS and PI3-Kinase signaling pathways, and is critical for processes such as wound healing and tissue regeneration (Grant, D. S. et al. (1993) Proc Natl Acad Sci USA 90: 1937-41; Watanabe, S. et al. (1994) Biochem Biophys Res Commun 199: 1453-60; Derman, M. P. et al. (1995) Am J Physiol 268: F1211-7; Bevan, D. et al. (2004) J Pathol 203: 831-8; Nakamura, T. et al. (2011) J Gastroenterol Hepatol 26 Suppl 1: 188-202). As a result, uncovering novel mechanisms for HGF-dependent activation of the Met receptor may provide new strategies for stimulating tissue repair in chronic wounds and fibrotic disorders (Nakamura, T. et al. (2011) J Gastroenterol Hepatol 26 Suppl 1: 188-202).
HGF is a secreted extracellular protein that exists as an inactive single-chain ligand (pro-HGF) until proteolytic cleavage at the Arg494-Val495 peptide bond results in a two-chain form, consisting of a disulfide-linked α/β-heterodimer, capable of activating the Met receptor (Nakamura, T. et al. (1989) Nature 342: 440-3; Naldini, L. et al. (1992) EMBO J 11: 4825-33; Shimomura, T. et al. (1995) Eur J Biochem 229: 257-61; Lee, S. L. et al. (2000) J Biol Chem 275: 36720-5; Peek, M. et al. (2002) J Biol Chem 277: 47804-9). The domain architecture of HGF is analogous to plasminogen, where the α-chain comprises an N-terminal PAN domain followed by four Kringle domain repeats (K1-4) and the β-chain contains the C-terminal trypsin/chymotrypsin-like serine protease domain (FIG. 1) (Donate, L. E. et al. (1994) Protein Sci 3: 2378-94; Tordai, H. et al. (1999) FEBS Lett 461: 63-7). Notably, both two-chain HGF and single-chain pro-HGF are capable of high affinity binding to the Met receptor through specific interactions with the α-chain; however, receptor activation can only occur after cleavage of pro-HGF into the two-chain form (Hartmann, G. et al. (1992) Proc Natl Acad Sci USA 89: 11574-8; Lokker, N. A. et al. (1992) EMBO J 11: 2503-10; Naldini, L. et al. (1992) EMBO J 11: 4825-33). Studies have indicated that during certain cases of liver cirrhosis and pulmonary fibrosis, the normal tissue repair process is severely compromised due to a lack of proteolytic conversion of available pro-HGF into the active form; leading to reduced Met signaling (Arakaki, N. et al. (1995) Hepatology 22: 1728-34; Kaibori, M. et al. (2002) J Surg Res 106: 108-14; Marchand-Adam, S. et al. (2006) Am J Respir Crit Care Med 174: 58-66; Phin, S. et al. (2010) Am J Respir Cell Mol Biol 42: 286-93). Not only does this highlight the importance of the cleavage step in regulating HGF-dependent Met signaling, but also suggests that allosteric activators that reversibly convert available pro-HGF into an active form, capable of Met signaling, would potentially yield a novel therapeutic approach to stimulating tissue repair in these indications (FIG. 2). There are many other indications where activation of HGF has potential benefit; for a review and partial list of tissues and disease areas see (Nakamura, T. et al. (2011) J Gastroenterol Hepatol 26 Suppl 1: 188-202) and Table 3 therein.
Extensive structural and biochemical work has revealed that HGF utilizes a trypsin/chymotrypsin-like serine protease activation mechanism for Met signaling (Kirchhofer, D. et al. (2004) J Biol Chem 279: 39915-24; Stamos, J. et al. (2004) EMBO J 23: 2325-35; Kirchhofer, D. et al. (2007) Proc Natl Acad Sci USA 104: 5306-11). Upon cleavage of pro-HGF, the newly formed N-terminus (Val495) in the serine protease-like β-chain, which corresponds to residue 16 in chymotrypsinogen numbering, inserts into a canonical ‘activation pocket’ (FIG. 3). Detailed work has shown that N-terminal insertion is critical for allosterically activating the β-chain, allowing for Met binding and subsequent activation of receptor signaling. Importantly, studies have shown that mutating the N-terminal Val495 to Gly or Asp672 to Asn within the β-chain prevents N-terminal insertion, thereby disrupting β-chain binding to Met and completely abolishing the signaling activity of two-chain HGF (Kirchhofer, D. et al. (2007) Proc Natl Acad Sci USA 104: 5306-11). Thus, the key mechanistic step for conversion of pro-HGF into a Met agonist is directly analogous to the activation of trypsin/chymotrypsin-like serine proteases (Khan, A. R. et al. (1998) Protein Sci 7: 815-36; Hedstrom, L. (2002) Chem Rev 102: 4501-24), despite the fact that the serine protease-like β-chain mediates protein-protein interactions rather than proteolytic activity.
Previously it has been shown that small peptides derived from the first 7-10 residues of the native N-terminus of HGF β target the serine protease-like activation pocket of a zymogen-like form of HGF β (scHGF β), allosterically activate Met binding, and subsequently activate a non-cleavable form of pro-HGF (scHGF) in cell-based Met signaling assays (Landgraf, K. E. et al. (2010) J Biol Chem 285: 40362-72). While this established the ability to allosterically regulate pro-HGF signaling activity by targeting the activation pocket of HGF β, the therapeutic potential of the approach was limited due to the very weak binding affinity of the activator peptides (KD˜2 mM) (Landgraf, K. E. et al. (2010) J Biol Chem 285: 40362-72). Therefore there remains a need for effective activators of HGF signaling.